FluoroSpot combines the sensitivity of ELISpot with the capacity to analyze secretion of several analytes simultaneously. This highly sensitive cellular assay is robust, easy to perform, and suitable for both single tests and large-scale screening. Mabtech provides FluoroSpot reagents for detection of a single analyte as well as dual, triple and quad FluoroSpot kits for combined detection of two, three or four analytes.

Triple FluoroSpot analysis enables studies of polyfunctional T-cell populations with different cytokine secretion profiles. In total, seven subpopulations can be visualized with triple FluoroSpot: three single secretors, three dual secretors, and one triple-secreting population. The images show a triple FluoroSpot analysis of IFN-γ (490), IL-2 (550), and TNF-α (640, color substituted) secretion by human PBMCs stimulated with a pool of viral peptides (CEF). Individual analyte images from the same well and an image overlay, combining images from the three filters, are shown.

• A fluorescence-based system enables simultaneous detection of multiple analytes

• FluoroSpot generates more data, saves time, and enables the use of fewer cells and less antigen compared to ELISpot

IFN-γ IL-2 TNF-α Overlay

Discover more with FluoroSpot

www.mabtech.com | mabtech@mabtech.com | +46 8 716 27 00 | Nacka Strand, Sweden

Coating A mixture of capture antibodies is added to all wells of an ethanol- treated PVDF membrane plate.

Cell incubation Cells are added in the presence or absence of stimuli and the plate is in-cubated to allow cytokine secretion.

Cytokine capture Secreted cytokines bind to the capture antibodies surrounding the activated cells.

BTAG

FITCCy3

Detection antibodies The cells are removed and a mixture of tag-labeled and biotinylated detection antibodies is added.

Fluorophore-labeled conjugates A mixture of fluorophore-labeled anti-tag antibody and streptavidin-fluorophore conjugate is added.

Analysis The plate is analyzed in a fluor- escence reader fitted with separate filters for the different fluorophores.

BTAG

FluoroSpot step-by-step guideStep-by-step guide to the FluoroSpot assay, demonstrated here by a dual FluoroSpot setup.

Discover more with FluoroSpotFluoroSpot can be used in numerous applications and is equally well suitable for single tests and large-scale screening. The sandwich assay design combined with fluorophore-labeled detection reagents enables analysis of two or more analytes in the same well.

A sandwich assay principle is applied in FluoroSpot in which a mixture of monoclonal capture antibodies with different specificities is coated onto PVDF membranes in a 96-well plate. Detection of dual-, triple- or quadruple-secreting cells is made possible by the use of a biotinylated detection antibody for one analyte and a tag-labeled detection antibody for the other analyte(s). The detection step is visualized and amplified by specific fluorophore-conjugated reagents and the resulting spots are analyzed in an automated reader.

FluoroSpot assay principle showing dual FluoroSpot. The use of fluorophores with maximum excitation at 380, 490, 550, and 640 nm requires a FluoroSpot reader equipped with DAPI, FITC, Cy3 and Cy5 equivalent filters, respectively.

SA-550

Detection mAb-biotin

Analyte 2

Capture mAb

Anti-tag mAb-490

Detection mAb-tag

Analyte 1

Capture mAb

TAG BFluorophore Filter

380 DAPI equivalent

490 FITC equivalent

550 Cy3 equivalent

640 Cy5 equivalent

T-cell FluoroSpotThe FluoroSpot assay is ideal for analysis of antigen-specific T-cell responses.

Delineating functional T-cell subsets by single-, dual-, and triple-cytokine profiling is of great interest in many different research fields. FluoroSpot can be used in vaccine development to detect and analyze vaccine-specific immmue responses by polyfunctional T cells. In cancer research, FluoroSpot can be used, for example, to monitor tumor-infiltrating lymphocytes or to provide a more direct assessment of cytotoxic T-cell responses by simultaneous analysis of IFN-γ and granzyme B secre-tion. We supply a wide range of FluoroSpot kits for analysis of single-, dual-, and triple-producing T cells where IFN-γ can be analyzed alone or in combination with several different analytes.

Human IFN-γ/IL-2 dual FluoroSpot The images show a dual FluoroSpot analysis of cells secreting IFN-γ and/or IL-2 in response to PPD (purified protein derivative). Of the 166 IFN-γ-secreting T cells, 114 also secreted IL-2. Dual-secreting cells were determined as spots with the same position (center point) in an image overlay of the 490 (IFN-γ) and 550 (IL-2) images.

B-cell FluoroSpotThe B-cell FluoroSpot assay can identify the presence of and quantify both the total number of anti-body-secreting cells and those secreting antigen-specific antibodies.

Detection based on fluorophores offers the possibility to analyze the secretion of different immunoglobulin isotypes or subclasses in the same well. Major application areas include analysis of B-cell responses in various diseases and those elicited by vaccination. For example, antigen-specific secretion of IgG, IgA, and IgM may be investigated before and after vaccination. Mabtech offers dual FluoroSpot kits for detection of human IgG/IgA and IgG/IgM and a triple FluoroSpot kit for simultaneous detection of IgG/IgA/IgM.

Human IgG/IgA/IgM triple FluoroSpot In the images shown, PBMCs were pre-incubated in the presence of R848 and recombinant human IL-2, added to a FluoroSpot plate, and incubated overnight. The number of cells secreting IgG (550), IgA (490) and IgM (640, color substituted) was analyzed.

230 spots 59 spots 75 spots

IgG IgA IgM Overlay

0 dual/triple spots

IFN-γ IL-2 Overlay

166 spots 157 spots 114 dual spots

FluoroSpot for other cell typesFluoroSpot can be used to analyze dendritic cells, monocytes, and macrophages.

Cytokine secretion by innate immune cells such as dendritic cells, monocytes, and macrophages can easily be studied using FluoroSpot. For instance, the assay can be useful for delineating distinct subpopulations based on their cytokine profile.

Human IL-6/TNF-α dual FluoroSpot The images show a dual FluoroSpot analysis of cells secreting IL-6 and/or TNF-α in response to the TLR7/8 ligand R848. Dual-secreting cells were confirmed as spots with the same position in an overlay of the 490 (IL-6) and 550 (TNF-α) images. Almost all IL-6-secreting cells also secreted TNF-α.

IL-6 TNF-α Overlay

335 spots 737 spots 304 dual spots

Mabtech develops and supplies FluoroSpot reagents for human, cow, monkey, and mouse research.

AnalysisAn automated reader with separate filters is needed for analysis.

The reader should be equipped with filters for excitation (ex) 490 nm/emission (em) 510 nm (FITC), ex 550 nm/em 570 nm (Cy3), ex 640 nm/em 660 nm (Cy5) and for four-color analysis also ex 380 nm/em 430 nm (DAPI).

Mabtech IRISTM FluoroSpot/ELISpot reader utilizes RAWspotTM technology for accurate identification of spot centers and spot numbers. In addition, it provides informa-tion on relative spot volume.

Mabtech IRISTM FluoroSpot reader.

PRODUCT SIZE CODE

Human FluoroSpot kits

Single FluoroSpot (Cy3 equivalent filter required)

Human GM-CSF FluoroSpotBASIC (550) for 4 plates FS1-15-550Human Granzyme B FluoroSpotBASIC (550) for 4 plates FS1-10-550Human IFN-γ FluoroSpotBASIC (550) for 4 plates FS1-01-550Human IL-1β FluoroSpotBASIC (550) for 4 plates FS1-12-550Human IL-2 FluoroSpotBASIC (550) for 4 plates FS1-02-550Human IL-5 FluoroSpotBASIC (550) for 4 plates FS1-08-550Human IL-6 FluoroSpotBASIC (550) for 4 plates FS1-13-550Human IL-8 (CXCL8) FluoroSpotBASIC (550) for 4 plates FS1-19-550Human IL-10 FluoroSpotBASIC (550) for 4 plates FS1-07-550Human IL-12/-23 (p40) FluoroSpotBASIC (550) for 4 plates FS1-14-550Human IL-13 FluoroSpotBASIC (550) for 4 plates FS1-04-550Human IL-17A FluoroSpotBASIC (550) for 4 plates FS1-03-550Human IL-22 FluoroSpotBASIC (550) for 4 plates FS1-18-550Human TNF-α FluoroSpotBASIC (550) for 4 plates FS1-09-550

Dual FluoroSpot (FITC and Cy3 equivalent filters required)

Human IL-1β/GM-CSF FluoroSpot kit 2 or 10 plates FS-1215-2/10Human IL-1β/IL-6 FluoroSpot kit 2 or 10 plates FS-1213-2/10Human IL-1β/ IL-12/-23 (p40) FluoroSpot kit 2 or 10 plates FS-1214-2/10Human IL-1β/TNF-α FluoroSpot kit 2 or 10 plates FS-1209-2/10Human IL-6/GM-CSF FluoroSpot kit 2 or 10 plates FS-1315-2/10Human IL-6/ IL-12/-23 (p40) FluoroSpot kit 2 or 10 plates FS-1314-2/10Human IL-6/TNF-α FluoroSpot kit 2 or 10 plates FS-1309-2/10Human IL-17A/IL-22 FluoroSpot kit 2 or 10 plates FS-0318-2/10Human IgG/IgA FluoroSpot kit 2 or 10 plates FS-05R06G-2/10Human IgG/IgM FluoroSpot kit 2 or 10 plates FS-05R17G-2/10Human IFN-γ/Granzyme B FluoroSpot kit 2 or 10 plates FS-0110-2/10Human IFN-γ/Granzyme B FluoroSpot kit, pre-coated 2 or 10 plates FSP-0110-2/10Human IFN-γ/IL-2 FluoroSpot kit 2 or 10 plates FS-0102-2/10Human IFN-γ/IL-2 FluoroSpot kit, pre-coated 2 or 10 plates FSP-0102-2/10Human IFN-γ/IL-5 FluoroSpot kit 2 or 10 plates FS-0108-2/10Human IFN-γ/IL-5 FluoroSpot kit, pre-coated 2 or 10 plates FSP-0108-2/10Human IFN-γ/IL-10 FluoroSpot kit 2 or 10 plates FS-0107-2/10Human IFN-γ/IL-10 FluoroSpot kit, pre-coated 2 or 10 plates FSP-0107-2/10Human IFN-γ/IL-13 FluoroSpot kit 2 or 10 plates FS-0104-2/10Human IFN-γ/IL-13 FluoroSpot kit, pre-coated 2 or 10 plates FSP-0104-2/10Human IFN-γ/IL-17A FluoroSpot kit 2 or 10 plates FS-0103-2/10Human IFN-γ/IL-22 FluoroSpot kit 2 or 10 plates FS-0118-2/10Human IFN-γ/TNF-α FluoroSpot kit, pre-coated 2 or 10 plates FSP-0109-2/10Human TNF-α/GM-CSF FluoroSpot kit 2 or 10 plates FS-0915-2/10Human TNF-α/ IL-12/-23 (p40) FluoroSpot kit 2 or 10 plates FS-0914-2/10

Triple FluoroSpot (FITC, Cy3 and Cy5 equivalent filters required)

Human IFN-γ/IL-10/IL-17A FluoroSpot kit, pre-coated 2 or 10 plates FSP-010703-2/10Human IFN-γ/IL-22/IL-17A FluoroSpot kit, pre-coated 2 or 10 plates FSP-011803-2/10Human IFN-γ/IL-2/TNF-α FluoroSpot kit, pre-coated 2 or 10 plates FSP-010209-2/10Human IFN-γ/IL-17A/IL-5 FluoroSpot kit, pre-coated 2 or 10 plates FSP-010308-2/10Human IFN-γ/IL-10/IL-5 FluoroSpot kit, pre-coated 2 or 10 plates FSP-010708-2/10Human IgG/IgA/IgM FluoroSpot kit 2 or 10 plates FS-050617-2/10

See the back for a list of monkey, cow and mouse FluoroSpot kits

www.mabtech.com | mabtech@mabtech.com | +46 8 716 27 00 | Nacka Strand, Sweden

We continuously expand our product portfolio. Please visit www.mabtech.com for a complete pricing and product listing.

PRODUCT SIZE CODE

Monkey FluoroSpot kits

Single FluoroSpot (Cy3 equivalent filter required)

Human GM-CSF* FluoroSpotBASIC (550) for 4 plates FS1-15-550Human IL-5* FluoroSpotBASIC (550) for 4 plates FS1-08-550Human IL-6* FluoroSpotBASIC (550) for 4 plates FS1-13-550Human IL-12/-23 (p40)* FluoroSpotBASIC (550) for 4 plates FS1-14-550Human TNF-α* FluoroSpotBASIC (550) for 4 plates FS1-09-550Monkey IFN-γ FluoroSpotBASIC (550) for 4 plates FS1-21-550Monkey IL-2 FluoroSpotBASIC (550) for 4 plates FS1-22-550

Dual FluoroSpot (FITC and Cy3 equivalent filters required)

Monkey IFN-γ/IL-2 FluoroSpot kit 2 or 10 plates FS-2122-2/10Monkey IFN-γ/IL-2 FluoroSpot kit, pre-coated 2 or 10 plates FSP-2122-2/10

Monkey IgG/IgA FluoroSpot kit 2 or 10 plates FS-05R24G-2/10Human IgG/IgM* FluoroSpot kit 2 or 10 plates FS-05R17G-2/10

Triple FluoroSpot (FITC, Cy3 and Cy5 equivalent filters required)

Monkey IgG/IgA/IgM FluoroSpot kit 2 or 10 plates FS-052417-2/10

* Reactivity with Human/Non-human primates

Mouse FluoroSpot kits

Single FluoroSpot (Cy3 equivalent filter required)

Mouse IFN-γ FluoroSpotBASIC (550) for 4 plates FS1-41-550Mouse IL-2 FluoroSpotBASIC (550) for 4 plates FS1-42-550Mouse IL-5 FluoroSpotBASIC (550) for 4 plates FS1-43-550Mouse IL-17A FluoroSpotBASIC (550) for 4 plates FS1-44-550

Dual FluoroSpot (FITC and Cy3 equivalent filters required)

Mouse IFN-γ/IL-2 FluoroSpot kit 2 or 10 plates FS-4142-2/10Mouse IFN-γ/IL-5 FluoroSpot kit 2 or 10 plates FS-4143-2/10Mouse IFN-γ/IL-17A FluoroSpot kit 2 or 10 plates FS-4144-2/10

Cow FluoroSpot kits

Single FluoroSpot (Cy3 equivalent filter required)

Bovine IFN-γ FluoroSpotBASIC (550) for 4 plates FS1-71-550Bovine IL-2 FluoroSpotBASIC (550) for 4 plates FS1-72-550

Dual FluoroSpot (FITC and Cy3 equivalent filters required)

Bovine IFN-γ/IL-2 FluoroSpot kit 2 or 10 plates FS-7172-2/10

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